The overall goal of this experiment is to study the in vivo gene expression dynamics of S tyrian bacteria growing inside tumors. First in a mouse xenograft cancer model, bacteria are injected intravenously by the tail vein. Bacteria colonize the tumor and grow exponentially while gene expression is visualized using luminescent reporters.
Whole animal bioluminescence imaging is performed using the IVUS spectrum imaging system. Over 24 to 48 hours a time course of IVUS images is taken. This image series is correlated with colony counts generated by homogenizing and plating tumors.
Together these data allow us to quantitatively measure the gene expression and population dynamics of tumor targeted bacteria in vivo in real time cell preparation passage, cell lines using standard cell culture techniques. In this experiment, we used ocar eight cells gross cells to a target co fluency of 80 to 100%Harvest and count cells on a hemo, cytometer, resus, suspend, and phenol red free DMEM at a target concentration of five times 10 to the seven cells per milliliter, or about 200 microliters per flask. Add 15%reduced growth factor matrigel and keep the cell suspension on ice until implantation tumor implantation.
Anes anesthetized four week old female nude mice using 3%isof fluorine. Load the cells 100 microliters per tumor in a one mill syringe and attach a 27 and a half gauge needle for each of two bilateral hind flank injection sites. Lift the skin gently with forceps to make a tent and inject cells at the base.
Take care not to penetrate too deep producing blood. Use tweezers to gently remove the syringe without spatter. Monitor tumor growth daily for 10 to 20 days until a tumor diameter of two to four millimeters is reached.
Bacteria preparation, start an overnight culture of bacteria in three mils LB media plus antibiotics from a minus 80 freezer stock or a refrigerated plate. In this experiment we used s tyer strain ELH four 30 in the morning. Dilute the culture one to 100 into filtered lb and grow to an OD 600 of 0.4 to 0.6.
Spin down and wash three times in phosphate buffered saline and resuspend at a final OD 600 of 0.1 or about one times 10 to the seven cells per mil. Bacteria injection, anesthetize animals, and position with the tail pointing toward your dominant hand. Locate the tail vein and dilate using warm water or a heat lamp.
Load the bacteria 100 microliters per mouse in a one mil syringe and bend the chip just less than 90 degrees. Align the syringe tip with the tail vein, penetrate at a shallow depth feeling no resistance and inject bacteria. Observe blood flow displacement for a successful injection.
Wait roughly 12 to 18 hours before imaging for colonization to occur. Mouse imaging anesthetize animals in the induction chamber. Then place each mouse on the imaging platform.
Maintain precise positioning to ensure quantitative results between time points, image animals using the Ibis Spectrum imaging system. If imaging more than one animal, place light barriers between them to prevent cross illumination animal dissection, holding the tumor with tweezers. Remove the skin using a reverse scissor motion.
Motion When the majority of the skin has been separated fully remove the tumor using tweezers. Place the organs and pre weighed micro centrifuge tubes. Since the mass of these tubes can vary significantly, it's important to pre weigh them for quantitative results.
Quantifying bacterial bio distribution for each time point excise and weigh the tumors add 500 microliters PBS plus 15%glycerol and homogenize using a tissue tear, generates serial dilution and plate for bacterial colony counting representative results. Using this protocol, we are able to generate data on the in vivo growth and gene expression dynamics of tumor targeted bacteria. The overall workflow is summarized here.
In the first stage, we injected bacteria and imaged the animal using ivus to measure gene expression with luminescence as a reporter. Then in the second stage, we excised, homogenized, and plated tumors for colony counts to determine the number of bacteria growing in the tumor. Bioluminescence generated by bacterial strains is visualized using ivus signal increases with injected dosage with a minimum colonizing dosage around five times 10 to the five in all cases due to attenuations.
The bacteria either specifically colonized tumors or fail to grow significantly in the mice signal is quantified using radiance units to normalize for exposure time or typical values above 10 to the six or greater are indicative of colonization for a given infection Signal increases with time over 24 to 48 hours. The peak onset is around 36 hours after injection here, but the timing may vary depending on the strain and plasmid being used. Be sure to position the mouse exactly the same way each time to ensure quantitative results in the next set of experiments.
Bacterial biodistribution is quantified to measure the growth of bacteria over time in different organs, viable bacteria and tumors are quantified by excising and homogenizing the tumor and plating its serial dilution. Other organs can be quantified. Similarly, here we have used spleen since the tumor spleen ratio is a typical measure of bacterial specificity after back calculating using the dilution factor.
These counts allow us to measure the specific bacterial population over time. On the left, we can see the number of bacteria grow steadily throughout the duration of the experiment. With a doubling time between one and three hours, substantially lower than in batch culture experiments.
This is likely due to the poor nutrient conditions in the tumor microenvironment. On the right, we have quantified the tumor spleen ratio and observed that it increases throughout the experiment. The goal of these experiments is to quantitatively monitor the dynamics of growth and gene expression of bacteria and tumor environments using IVUS imaging and colony counting techniques.
We have shown that populations of bacteria can be characterized in vivo in tumor environments. In the future, more complex gene expression programs can be designed and engineered to build sophisticated therapeutic delivery systems in bacteria.I.
